During our transthoracic echocardiography (TTE) courses, medical students showed difficulty in spatial orientation. We implemented the use of 3D printed cardiac models of standard TTE views PLAX, PSAX, and A4C and assessed their efficacy in TTE-teaching.n = 77) was taught using 3D models; the control group (n = 76) without. Both were comparable with respect to baseline parameters. Besides test-scores, a Likert scale recorded experiences, difficulties, and evaluation of teaching instruments.One hundred fifty-three participants were split into two groups. A pre-test-retest of anatomy, 2D -, and 3D orientation was conducted. The intervention group (p < 0.001). However, the intervention group taught with the 3D models, scored significantly better overall (p = 0.016), and in 2D-thinking (p = 0.002) and visual thinking (p = 0.006) subtests. A backward multivariate linear regression model revealed that the 3D models are a strong individual predictor of an excellent visual thinking score. In addition, our study showed that students with difficulty in visual thinking benefited considerably from the 3D models.From the 153 students evaluated, 123 improved, 20 did worse, and ten achieved the same result after the course. The median overall pre-test score was 29 of 41 points, and the retest score was 35  performed significantly better than the control group (n = 76).• The 3D group (• We advocate 3D printed models for the teaching of echocardiography standard views. Transthoracic echocardiography (TTE) is an essential clinical skill which is a routine part of cardiac surgery and cardiology resident training. However, also medical students often express an interest in this topic and request voluntary hands-on courses. Echocardiography offers an enhanced understanding of cardiac anatomy and physiology. Accordingly, the new German national competence based catalogue of learning objectives (NKLM) for medical schools suggests sonography as a skill, students should be exposed to during their studies .Upon this request, we offered our first voluntary echocardiography curriculum in 2017; and the course proved to be very popular. The content of our course adhered to the CME certified education “Basic transthoracic echocardiographic skills in cardiac surgery patients ” and folDuring the first 3 years of the program, students frequently reported problems in spatial intracardial orientation, which hampered image interpretation and the learning progress. To overcome this by didactical means, 3D printed cardiac models were introduced to this course.Didactic advantages for the use of 3D models have been described in anatomical teaching , additioDue to changes of the curriculum secondary to the COVID-19 restrictions, our voluntary course became obligatory for an entire semester cohort following strict hygienic standards. This allowed for us to cross-sectionally teach and study an entire semester-cohort in a highly standardized manner without bias to previous knowledge or special interests in this topic. Complementarily, the department of internal medicine has established an complementary sonographic curriculum .The objective of this project was to study the didactical impact of 3D printed models of standard echocardiographic views on the students’ learning success during a teaching intervention in a controlled trial.The study was designed as a unicentral, prospective, controlled, unblinded, cross-sectional trial, extensively comparing two equally sized groups of medical students of a complete, unselected semester-cohort for learning success in TTE with or without the usage of 3D models. Participation in the study was voluntary and subjects submitted their written informed consent. We enrolled 195 medical students in their 9th semester.The educational objective was to learn, understand, and reproduce three basic standard views in TTE. Five online resources flanked the 120- min front-of-class lecture according to Fig. The second online resource gathered baseline-information: participation in previous sonography courses, being a tutor-student in sonography, and previously visited preclinical classes on cardiac anatomy. Additionally, a five-point Likert scale recorded their self-assessment concerning their experience in sonography, echocardiography, and cardiac anatomy.The students participated in the 120-min echocardiography class within the adjacent week. Groups of up to eight students started the class with the third online resource called the ‘ex-ante’ pre-test. The test showed artificial pictures of three selected echocardiographic standard views , 2D orientation, 3D orientation, and each domain was represented by multiple questions. The questions demanded free-text answers for more discriminative power than multiple-choice questions.We used open questions demanding free text answers due to the reasons described by Loke et al. . Open quVoluntary pairs examined each other with the four echocardiography devices . The tutor occasionally guided and corrected each student pair until they could reproduce the three standard views: apical four-chamber view (A4C), parasternal long-axis view (PLAX), and parasternal short-axis view (PSAX). Thereafter, the participants heard a short lecture on common echocardiographic findings in emergency medicine. Then the participants performed the identical retest. Finally, they filled in an online questionnaire, asking them about their perception on the helpfulness of teaching means and to rate the difficulty of solving the tests.https://www.resuscitativetee.com/3d-printed-models) . The distribution of the individual differences between pre- and retest scores in the control and intervention groups fulfilled criteria of normality for the overall test results but not concerning the subdomains  of the test. We performed a multivariate linear regression with ‘improvement in the visual thinking score’ as the dependent variable. The visual thinking score comprises the subdomains 2D and 3D orientation and was formed by adding the respective points from the questionnaire. The independent variables were ‘age’, ‘gender’, ‘3D model intervention’, ‘abdominal sonography class’, ‘score in the hose-figure test’, and ‘visual thinking pre-test score’. These potential explanatory variables were chosen based on expert opinion and reasoning without statistical preselection. An analysis of variance (ANOVA) was carried out for self-assessment and test results. Only complete data sets were admitted for statistical analysis. The evaluation strategy was supported by the Institute of Clinical Epidemiology and Applied Biometry of the University of Tübingen. Statistical analysis and data reporting complied with the EACTS statistical and data reporting guidelines [We aimed to include half the students in the 3D model intervention group and half in the control group. All available cases were analyzed with SPSS 25 . Continuous variables were tested for normal distribution by the Shapiro-Wilk test. Normally distributed data are reported as means and standard deviations (SD); otherwise, the medians, quartiles and ranges are reported. Categorical data are reported as counts and percentages. For inferential statistical comparisons of categorical variables, the idelines .From the 195 students evaluated, 164 gave written informed consent. The others attended the class, but no data was recorded. 153 (100%) students returned valid results (11 datasets had to be excluded due to incomplete answers). 89 (58.17%) students were female. The mean age was 25.9 +/− 2.9 years. Experience in sonography was as follows: 72.5% (111) attended the abdominal sonography class, 9.2% (14) were peer-teaching tutors, 1.96% (3) attended the preclinical heart study group, and 11.1% (17) had clinical experience. Just 12.4% (19) reported having no practical experience. On a Likert-scale from 1 (poor) to 5 (excellent) points, the participants had to self-assess their expertise before the class (Table n = 76) and the intervention group (n = 77) did not differ significantly with respect to age, gender, experience, and self-assessment. The majority of the control group patients  had already participated in an abdomen-sonography class, whereas this was only true for 44/77 (57%) of the patients in the intervention group .The control group (up Table . There wp < 0.001). Both groups improved significantly. In the control group, subjects improved by a mean of 4.6 ± 6.5 points. In the 3D model intervention group, the mean improvement was significantly greater   and visual thinking (p = 0.006), and a strong tendency with respect to 3D orientation (p = 0.088)  Fig. .A qualitative observation from the course: Students claimed the models largely contributed to their understanding. In addition, they often requested them for their learning sessions and gave positive reviews.1 2.96, CI 95% 1.487–4.44, p < 0.001), a good visual thinking pre-test score , and participation in the concomitant abdominal sonography class . The model achieved a corrected R2 of 0.404. The effect size according to Cohen was f2 = 0.62. Importantly, teaching with the 3D models revealed to have the strongest influence on the increment of post-test visual thinking. The students, on average, scored 2.96 points more when being taught using the 3D models.A backward multivariate linear regression model sought to identify independent variables that predict an improvement from pre- to retest. From the candidate independent variables age and gender were eliminated as irrelevant. Likewise, the test score in the hose-figure test was not predictive Table . Predictp = 0.001). The same effect was present for anatomy questions  and 2D-thinking questions , respectively. In summary, in all three questionnaire categories, student self-assessment was related to performance. This was not the case for performance in 3D-thinking questions.The students completed a self-assessment regarding the difficulty of the questions in the pre-test with a 5-point Likert scale. Later the 5 point scale was aggregated into three categories  and compared with the objective test results. Students with subjective difficulty in the hose figure test, objectively scored only 75% (CI 95% 63.25–86.75) whereas students rating it easy scored 92% .Anatomical models are traditionally available for a more in-depth understanding of the topology of organs, and haptic models transport this concept better than 2D images or virtual reality computer models , 16. 3D TTE is not part of the core curriculum of medical students in Germany, but it is suggested by the national competence based catalogue of learning objectives in medicine . EchocarThe central findings of this study are: (a) the intervention group taught with the use of 3D models scored significantly better overall, and in terms of visual thinking, and (b) the 3D models have the most substantial positive influence (effect size) on the post-test results.Self-assessment and test scores showed that a higher perception of difficulty coincided with a lower test score, which is conclusive. Especially students with below-average pre-test results in visual thinking benefit considerably from the 3D models. Students rated the learning experience very positively. Thus, teaching with haptic 3D-models subjectively and objectively leads to more effective teaching and learning in echocardiography classes.With this, we provide evidence for the superiority of the use of three-dimensional models compared to conventional imaging-based teaching materials in echocardiographic training and strengthen the basis for further systematic teaching research in the field. The strategy may be transferred to other sonography classes, especially given the fact that the 3D printing technology enables creating models tailored to the suits of the students and according to the didactic requirements in a cost effective and timely manner.There were no formal sample size calculation prior to the study, we cross-sectionally analyzed an entire unselected semester cohort instead. The number of test-questions to assess each level of understanding  was limited to three each, which might be discussed as relatively few, and the teaching intervention itself was restricted to a one-day session, which is critically short. On the other hand, the relatively large number of participants may compensate for some of these shortcomings.In the same semester, our colleagues from internal medicine performed an abdominal sonography class for the same cohort . To restOtherwise, the baseline characteristics of the groups did not differ . The three-dimensional anatomical orientation of the echo views was subject of pre- and retests. This strategy of using the same test twice contains a learning bias, which is controlled by exposing both groups to that strategy. Larger longitudinal studies are warranted to compensate for the aforementioned limitations.The implementation of 3D printed models of the standard echocardiographic views PSAX, PLAX, and A4C lead to a significantly more effective training in echocardiography, especially regarding the transfer of visual-thinking-skills. Particularly students with below-average performance in visual thinking benefited from this intervention. This effect may be transferable to other teaching interventions and must be object to future studies in evidence-based medical- and surgical education.


Since 2014, sleeve gastrectomy (SG) has been the most frequently performed bariatric-metabolic operation worldwide . There are only a few studies reporting a long-term follow-up (up to 11 years) available today. The aim of this study was to evaluate the long-term outcome of SG with a follow-up of at least 15 years regarding weight loss, remission of associated medical problems (AMP), conversions, and quality of life (QOL).Multicenter cross-sectional study; university hospital.This study includes all patients who had SG before 2005 at the participating bariatric centers. History of weight, AMP, conversions, and QOL were evaluated by interview at our bariatric center.2. Twenty-six patients (49.1%) were converted to Roux-en-Y gastric bypass (RYGB) for weight regain and gastroesophageal reflux within the follow-up period. Total weight loss after 15 years was 31.5% in the non-converted group and 32.9% in the converted group. Remission rates of AMP and QOL were stable over the follow-up period.Fifty-three patients met the inclusion criteria of a minimal follow-up of 15 years. Weight and body mass index at the time of the SG were 136.8kg and 48.7kg/mFifteen years after SG, a stable postoperative weight was observed at the cost of a high conversion rate. Patients converted to RYGB were able to achieve further weight loss and preserve good remission rates of AMP. SG in patients without the need of a conversion to another bariatric-metabolic procedure may be considered effective. Careful preoperative patient selection is mandatory when performing SG. Obesity is a disease that continually increases in numbers worldwide. It is strongly associated with numerous medical problems of the metabolic syndrome . Today, 2) as the first step of a two-step procedure. A second bariatric-metabolic procedure would be performed after initial weight loss [In its beginning, SG, a mainly restrictive operation which entails the resection of a greater part of the stomach, was a component of biliopancreatic diversion with duodenal switch . Later, ght loss . Currentght loss .Studies reporting a short-term follow-up after SG usually present good weight loss, remission of associated medical problems (AMP), and only low numbers of patients with weight regain (WR) and gastroesophageal reflux disease (GERD) . In studThe efficacy of a bariatric-metabolic procedure may only truly be evaluated at a long-term follow-up. However, there are just a few studies reporting a long-term follow-up up to 11 years) available in the literature today  years av. Thus, tAll patients that underwent SG for obesity before December 2005 in one of three Austrian bariatric centers were included in this multicenter study. The participating centers were Medical University of Vienna, Hospital Rudolfstiftung in Vienna, and Hospital Klosterneuburg.This study was approved by the Review Board of the ethical committee of Medical University of Vienna (Reference number: 2169/2019). Informed consent was obtained from all participating patients.The included collective represents a 15-year follow-up of the same 53 patients reported by Felsenreich et al. in 2016 (10-year follow-up after SG) . Each paPatients who still have their SG after 15 years (non-converted patients), as well as patients who were converted within the follow-up period, were interviewed on their history of weight, remission of AMP, and conversions to other bariatric-metabolic procedures. Additionally, non-converted patients were asked to complete the following questionnaires on the outcome after bariatric-metabolic surgery and QOL: Bariatric Analysis and Reporting Outcome System (BAROS) , BariatrTo achieve a high follow-up rate, patients were contacted per telephone or mail and called in for an interview. In Austria, the current home address can be acquired from the registration offices for scientific reasons as well as data from the obituary column to avoid patients lost to follow-up. Data of deceased and acutely converted patients (within a short period of time after the SG) were included in the baseline characteristics but not in the further calculation of the follow-up.The technique of performing a SG Fig.  was simiThe technique of performing a SG common then has only changed slightly up until now. The differences are that today, we use a slightly smaller bougie size of 12mm (36 French) and start the resection slightly closer to the antrum (2–3cm from the pylorus). The complete resection of the entire fundus was performed then and is still done today .Weight and BMI (body mass Index) at the time of the SG (as well as at the time of the conversion in converted patients) were gathered from operation protocols. Patients were asked for their nadir weight (including point in time when it was reached) after SG . Data on patients’ 10-year postoperative weight was published in the above-mentioned publication for the same cohort . As befoAll operation protocols and electronic patients’ charts were studied to gather information about the reason for the conversion, body weight, status of AMP, etc.The status of AMP at the time of the SG (as well as at the time of the conversion in converted patients) was gathered from the operation protocols. The status of AMP at 10 years postoperative was published before , and forThe outcome of the SG and the QOL at the long-term follow-up were both evaluated using questionnaires  only in non-converted patients.BAROS is a validated tool to evaluate the outcome of bariatric-metabolic surgery. It consists of five categories  the outcome of which is classified as failure, fair, good, very good, or excellent .The BQL is a user-friendly questionnaire to gather information about patients’ QOL after bariatric-metabolic surgery. It consists of 13 questions, with a maximum total score of 65 points. It creates a comprehensive picture of patients’ QOL in their daily routine , 16.SF-36 is a validated general QOL score including 36 questions that represent patients’ QOL in 8 different categories that focus on physical , physical role (RP), bodily pain (BP), general health (GH)) and mental QOL , mental health (MH), vitality (VT), social functioning (SF)) .2 . The number of participants included in this study was limited by the number of patients that were able to reach the follow-up of at least 15 years. Data was collected in Excel for Windows , and statistical calculations were performed using SPSS V24 for Windows .The presentation of data in this study was as percentage, by mean and standard deviation (SD), or median and range (R). Comparing different groups of data, either non-parametric Mann-Whitney U test or chi-square test was used. Univariate analysis was two-tailed with statistical significance defined as a p-value <0.05. Excess weight loss (EWL) in % was calculated based on a BMI of 25kg/mFifty-three patients received SG as a bariatric-metabolic procedure before December 2005 in any of the three participating centers, and, therefore, the follow-up of this study is at least 180 months. The patients’ characteristics are highlighted in Table Four patients died within the 15-year follow-up period; however, their deaths were not associated with any bariatric-metabolic surgical procedure. These four patients and one patient who was acutely converted for early leak were excluded from further analysis in terms of weight loss and improvement of AMP. Another acutely converted patient, who is one of the four deceased patients, died more than 12 years after the conversion.A complete follow-up after 15 years was attained in 46 of the remaining 48 patients (95.8%). The median follow-up period was 186 months (range 181–207 months) ≙15.5 years.A total of 26 49.1%) of 53 patients were converted: 25 to Roux-en-Y gastric bypass (RYGB) and one to biliopancreatic diversion with duodenal switch. The main indication for the conversion was WR in 14 patients (26.4%), symptomatic GERD in ten patients (18.9%), and acute conversion for an early leak in two patients (3.8%); see Table % of 53 p2. The history of weight within the 15-year follow-up period, excluding deceased and acutely converted patients, is presented in Table 2 and 30.9 ±6.2kg/m2, representing a total weight loss (TWL) of 36.9 ±11.7.Mean weight and BMI at the time of the SG were 136.8 ±27.8kg and 48.7 ±9.2kg/m2 after a median period of 48 months. After a follow-up of 186 months, the non-converted group was able to reach a weight, BMI, and TWL of 93.0 ±22.1kg/m2, 34.3 ±6.9kg/m2, and 31.5 ±12.6%. The weight, BMI, and TWL of the converted group was 91.6 ±19.3kg/m2, 31.5 ±5.5kg/m2, and 32.9 ±15.1%. A detailed history of weight in patients converted mainly due to reflux or weight regain is highlighted in Table Weight and BMI at the time of the conversion in the entire group of converted patients were 112.0 ±24.6kg and 38.4 ±6.2kg/mThe history of AMP, including improvement and new onset, is shown in Table New diagnoses of AH, DMII, HL, OSA, and DBJ within the 15 years of follow-up were observed in 5.6%, 2.1%, 2.2%, 0%, and 4.4%, respectively.Outcome scores and QOL of non-converted patients after 15 years are highlighted in Table This multicenter study presents a follow-up of at least 15 years after SG in terms of weight loss, conversion rate, remission of AMP, QOL, and data from bariatric-metabolic outcome scores. To the authors’ best knowledge, this study represents the longest follow-up after SG in the literature so far with a high follow-up rate. As mentioned earlier, this study is an update of the exact same patient collective published 5 years ago, which presented a follow-up of 10 years after SG . TherefoData on a long-term outcome of more than 10 years focusing on weight loss after SG are rare in the literature. However, comparing this study’s data (15 years follow-up) to the previous study with a 10-year follow-up, an interesting trend may be observed. Long-term weight loss remains stable or even decreases, however, does so at the cost of a higher conversion rate. To be exact, mean weight of the total collective decreases from 98.1 (after 10 years) to 92.4kg (after 15 years), whereas the conversion rate increases from 35.8 (after 10 years) to 49.1%. This shift of patients with weight regain from the non-converted group to the converted group with further weight loss leads to better results in both groups. Also, the mean EWL of the total collective increases from 54.0 (after 10 years) to 61.0% (after 15 years). The high number of additional conversions within the last 5 years was, in a manner of speaking, in part caused by our previous study . As patiThere is a small number of studies on SG with a follow-up of at least 10 years available in the literature today. Chang D.M. et al. presented 65 patients after SG that reached a long-term follow-up of 10 years. The authors report an EWL of 70.5% with a low conversion rate of only 16.9%; however, the follow-up rate was only 64.4%  and a veIt may be summarized that long-term studies after SG showed a relatively stable weight and acceptable weight loss rate, and an increasing conversion rate over time.The current study presents the first remission rates and rates of new onsets of AMP at more than 15 years after SG. Nevertheless, the description is based on a relatively small population as only 29.5% of the patients had at least one associated medical problem at the time of the SG. Therefore, the validity of the data based on AMP is limited.Regardless of the small population, the improvement rates are relatively stable over time with only small numbers of new onsets compared to our 10-year outcome study . This haAlso, the remission rates of AMP after SG in other long term-studies with a follow-up of more than 10 years are based on small patient populations as well. In terms of DMII remission, rates between 39.1 by Chang D.M. et al.  after 10It can be summarized that acceptable to good remission rates of AMP after SG still exist after a long follow-up period of 10–15 years. Nevertheless, these outcomes are based on rather weak data due to small study cohorts. For more reliable data, the publication of randomized control trials (SM-BOSS and SLEEVEPASS) on long-term outcomes after SG has to be awaited—they currently report mid-term outcomes .The following QOL and outcome scores were used in the current study: BAROS as an outcome score after bariatric-metabolic procedures, BQL as a QOL questionnaire specifically created for patients with obesity after a bariatric-metabolic procedure, and SF-36 as a general QOL questionnaire reflecting a bigger picture of patients’ psychological and physical QOL.A comparison of the BAROS score in today’s study to the data gained from the score in our previously published study 10 years after SG shows that this score has increased from 2.4 (10 years) to 2.9 (15 years) in non-converted patients after SG; nevertheless, the classification is still the same reflecting a fairly efficient outcome. The reason for the increased score is simply based on the high conversion rate, as patients converted due to weight regain were not included in this analysis and weight loss has a major impact on this score. A recently published study by Fiorani C. et al. reported a BAROS of 1.47 at an 8-year follow-up after SG .Comparing data gained from the BQL and SF-36 scores in this study and our 10-year follow-up shows a slight improvement from 10 to 15 years after SG in the BQL as well as all eight categories of the SF-36 . Again, A recent study by Major P. et al. reports SF-36 data in 28 SG patients before the operation, at 1 year, and at 10 years after . All 8 cTo conclude, the best way to interpret patients’ QOL is comparing one patient collective’s data from different points in time, thus taking a longitudinal perspective. All the reported scores proved valid to work out the individual patient’s QOL at different points in time after SG.This study presents a small collective from the beginning of SG as stand-alone procedure. The common operation technique has slightly changed since then, even though a worldwide standard does not exist, yet. To increase the number of patients, patients from other bariatric centers with the same operation technique at the time were included; however, the number of patients may still be too small to draw any final conclusions. Additionally, this also increases the number of surgeons who performed the procedure in these patients.Another limitation is that 10 patients had gastric banding before SG, which may have affected weight loss and outcome. However, it should be considered that patients with previous bariatric-metabolic procedures reflect the typical collective of patients bariatric surgeons deal with. Also, the size of the bougie used in SG has changed slightly, which may lead to different weight loss outcomes in recently operated SG patients.Finally, the current study lacks preoperative data on QOL; therefore, the collected data cannot be compared to patients’ QOL before SG.Fifteen years after SG, a good and stable postoperative weight was observed but at the cost of a high conversion rate of up to 49.1% due to weight regain and reflux. Patients converted to RYGB were able to achieve further weight loss and preserve good remission rates of AMP. SG in patients without the need of a conversion to another bariatric-metabolic procedure may be considered effective. In conclusion, a careful preoperative patient selection is mandatory when performing SG in patients with obesity.


D3h and Cs symmetry are synthesized by Vollhardt trimerization of C2v-symmetric dumbbell structures with central acetylene units and subsequent intramolecular ring closure. Scanning tunneling microscopy of the D3h-symmetric species at the solid/liquid interface on graphite reveals triporous chiral honeycomb nanopatterns in which the alkoxy side chains dominate the packing over the carboxylic acid groups, which remain unpaired. In contrast, Cs-symmetric isomers partially allow for pairing of the carboxylic acids, which therefore act as a probe for the reduced alkoxy chain nanopattern stabilization. This observation also reflects the adsorbate substrate symmetry mismatch, which leads to an increase of nanopattern complexity and unexpected templating of alkoxy side chains along the graphite armchair directions. State-of-the-art GFN-FF calculations confirm the overall structure of this packing and attribute the unusual side-chain orientation to a steric constraint in a confined environment. These calculations go far beyond conventional simple space-filling models and are therefore particularly suitable for this special case of molecular packing.Molecular spoked wheels with  D3h and Cs symmetries on graphite show the competitive or complementary effects of carboxylic acid groups and alkoxy chains on the nanopattern formation.Scanning tunneling microscopy investigations of phenylene-based molecular spoked wheels with  They adsorb along the HOPG main axis (i.e. zig–zag) directions,13 and only few exceptions are reported for rotated domains in constrained environments, which are then oriented along the armchair direction, whereas other random orientations have not been observed.14 Disk-shaped molecular backbones of a certain size and shape generally require an adequate alkyl/alkoxy proportion in order to keep them soluble, and 3,4,5-trihexadecyloxybenzyloxy groups have been proven as successful substituents for this task as well as for the physisorption of shape-persistent macrocycles (SPMs) and MSWs on HOPG.10 In the latter case, the formation of D6h-symmetric structures is a direct consequence of the synthetic strategies in which either six equal spoke-rim-segments are coupled to a central (arylene) hub (via Sonogashira reaction),10 or symmetrical acetylenes are trimerized (via Vollhardt cyclization)10 before the rim is closed. While efforts have been made to study how the symmetry of 2D supramolecular tectons is related to the nanopatterns formed,16,17 reducing the symmetry of the MSWs has not yet been described. Vollhardt acetylene trimerization of an unsymmetrical acetylene is a straightforward method to obtain such compounds.Carbon-based structures composed of arylene, arylene–ethynylene, and arylene–butadiynylene units have become prominent target structures in basic research with broad applications in materials as well as medical chemistry,11 and also trimellitic acid 18 are available and allow a comparison between the packing behavior of the respective esters and acids. The latter generally dimerize intermolecularly at the surface and stabilize the adsorbate packing. However, it is not predictable if the bulky flexible side groups allow this stabilization also in the present case or if instead the alkyl packing dominates the pattern formation.When one of the acetylene ends contains the aforementioned 3,4,5-trihexadecyloxybenzyloxy unit (bulky corner in D3h- and Cs-symmetric MSWs 1a/b and 2a/b (Rf) as compared to the 1,3,5-isomer, in which the different polarities of the ester groups are cancelled out. Final ring closure of both species was performed under Yamamoto coupling conditions, and the products were obtained as D3h-symmetric  and Cs-symmetric  methyl esters 1a and 2a, from which the carboxylic acids 1b and 2b were obtained.Here, we report on the synthesis of and 2a/b  and the and 2a/b . These w1a in dichloromethane, were fully characterized in solution by NMR and PL lifetime measurements . However, a final proof of the structure of the wheels 1a/b and 2a/b is obtained by scanning tunneling microscopy  solution using octanoic acid (OA) as a solvent. The choice of solvent was optimized in thorough previous work on different MSWs.20 Initial studies on 1a in OA  nm and γ = (60 ± 2)° containing two molecules with identical conformations but different (relative) orientations cA and cB with γ = 180° dimers, in contrast to trimesic acid11 and its larger homologues.11 The functional group distances  nm, b = (5.7 ± 0.2) nm, γ = (88 ± 2)° containing two molecules (with backbones rotated by 180°) is indexed. Of each pair of molecules, 14 of the 18 hexadecyloxy side chains (shown in bright and dark blue color in 2a (shown in purple color in 13 domains of (cyclo-) alkanes oriented along the armchair direction in a constrained environment have been observed previously,14 although this arrangement is rather rare. In addition, in the STM images medium-bright features similar in shape and length to those of the alkoxy side chains are clearly visible, which cannot be attributed to MSW entities. As in the case of earlier results, OA (i.e. solvent) molecules form dimers on the surface, which fill the otherwise uncovered space on the surface, align in parallel to the (MSW) alkoxy chains, and therefore stabilize the packing. As such, we suspect that the alkoxy chains aligned along the less favorable armchair direction are stabilized by two intercalating solvent dimers, which are clearly visible in the STM images  on a graphene cutout (C5644H190) containing the relevant monolayer regions , which would be prohibitively expensive using even fast semi-empirical quantum mechanical tight-binding calculations (e.g. GFN2-xTB) due to the number of atoms involved. Additional details of the calculations are found in the ESIcf.28 More precisely, after geometry optimization of the aforementioned starting geometries by GFN-FF, the alkoxy side chains in 13 Moreover, hydrogen bond dimerization of the OA carboxylic acid groups is also retained during optimization. Both these results resemble the high degree of descriptiveness of non-covalent interactions by the GFN-FF method. A more detailed discussion of this issue is given in the ESI.‡To scrutinize this assumption, for this highly specific question we performed geometry optimizations of four MSWs 1a, the ester groups in 2a do not interact, and are D4 = 2.3 nm apart  nm is indexed, however, neglecting different backbone orientations. According to this packing distance along a, we assume that only 10 of the 12 side chains along arrow 12  is attributed to the reduced order owing to the aforementioned factors. However, each molecule determines the orientation of the other molecule  by side-chain interactions (arrows 11 and 13), whereas the orientation of the molecules in the subsequent row is not influenced (arrows 12 and 14). Occasionally, hydrogen-bonded trimers are observed  form self-assembled monolayers on HOPG, and were studied by STM with submolecular resolution. These investigations show that the D3h-symmetric 1,3,5-isomer forms the same packing regardless of the exact carboxylic group substitution (i.e. ester or acid), implying that the 2D crystal structures are dominated by the hexadecyloxy side chains. Contrary, the packing of the Cs-symmetric 1,2,4-isomer is strongly influenced by the absence or presence of substituents prone to forming hydrogen-bonded dimers. Alkoxy side chains play only a subordinate role so that in 2b the superstructure formation is driven by dimerization of the carboxylic acids. The monolayer of 2a acts as a template for the armchair-oriented adsorption of alkyl chains, including OA solvent molecules. A complex computational analysis by means of GFN-FF could confirm this rather unusual STM observation and demonstrates the potential of the method to go far beyond the conventional level of STM image description using space-filling models.In summary, we described low-symmetry MSWs with ester groups in the 1,3,5- or 1,2,4-positions, respectively, which upon hydrolysis give the corresponding free acids that can be viewed as giant alkoxy-substituted homologues of trimesic and trimellitic acids. Besides spectroscopic, spectrometric, and chromatographic methods, STM is an integral part of the structure assignment. All four MSWs (S. H. elaborated the research concept and acquired funding. C. S. and T. L. S. synthesized, isolated and characterized the compounds and studied aggregation of the MSWs. T. J. K. and J. B. performed STM investigations and modelling of MSW nanopatterns and together with S.-S. J. visualized and curated the data. M. B., J. K., and S. G. performed theoretical investigations incl. design of computer programs, code implementation and/or testing as well as data visualization and analysis. T. E. performed optical spectroscopic investigations. J. M. L., S. G., S. H., and S.-S. J. administrated the project incl. supervision and validation of the results. The original draft was written through contributions of all authors. T. J. K., J. B., S. H., and S.-S. J. revised and edited the final manuscript.There are no conflicts to declare.SC-012-D1SC01381E-s001


Role of interleukin 17A (IL-17A) in carcinogenesis and cancer growth is controversial. Although some researches support its antitumor activity, some others suggest that it promotes the growth and development of different types of cancer including skin cancer by activation of STAT3. Although the function of the cytokines such as IL-17A has been extensively studied in various types of cancer, nonmelanoma skin cancer (NMSC) has not received much attention. Therefore, the present study was aimed to investigate the serum levels of IL-17A in NMSC patients.  This cross-sectional study was performed on 60 patients with basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) as well as 57 age-sex matched healthy individuals as control group. Measurement of IL-17A serum levels in both case and control groups was performed by a commercially reliable sandwich enzyme-linked immunosorbent assay (ELISA) kit. P < 0.001). Also, both BCC and SCC patients had higher levels of IL-17A in their sera in comparison to the controls . However, there was no significant difference between SCC and BCC patients regarding serum levels of IL-17A.  In this study, we observed that IL-17A serum levels in NMSC patients were significantly higher than the control group ( According to our results, it can be concluded that IL-17A may play a role in inducing the growth and progression of NMSC and it can be used as a therapeutic target in these patients in future. Skin cancer is one of the substantial health problems, which is the most common and most preventable malignancy in the world and its incidence does not show any signs of plateauing . Skin caγδT cells, and natural killer cells can secrete IL-17A [α (TNF-α), granulocyte colony-stimulating factor (G-CSF), chemokines, matrix metalloproteinase (MMP) 13, receptor activator of nuclear factor kappa-B ligand (RANKL), and antimicrobial peptides  [The interleukin 17A (IL-17A), previously known as cytotoxic T lymphocyte-associated antigen 8 (CTLA-8), is a T cell-derived proinflammatory cytokine which was introduced by Rouvier et al. in 1993 [e IL-17A . IL-17A e IL-17A . IL-17A 100A8/9) .Although some researches support antitumor activity of IL-17A, some others suggest that it promotes the growth and development of various types of cancer including skin cancer . Due to This cross-sectional study was performed on 60 patients with BCC and SCC skin cancers. They were recruited from dermatology clinic of Faghihi Hospital affiliated with Shiraz University of Medical Sciences who had no previous history of treatment including surgery and anticancer drug or radiation therapy, as well as 57 age-sex matched healthy individuals as the control group without any history or evidence of cancer, autoimmune and genetic disorders in themselves, and their first-degree relatives and had no inflammatory or infectious diseases at least for the past 3 months. Approval for the study was obtained from the Ethical Committee of Shiraz University of Medical Sciences (IR.sums.med.rec.1397.163) and all patients and control group gave written informed consent before participation in the study. Demographic, medical history and clinical data were collected from medical documents or clinical examinations.5 ml of the blood sample was obtained from all participants; the sera were separated and stored at −80°C until analysis. Measurement of IL-17A serum levels in both case and control groups was performed by a commercially reliable sandwich ELISA kit  according to manufacturer's protocols and using the standard samples with known levels of IL-17A, provided by the manufacturer and presented as pg/ml.P values of 0.05 or less were considered statistically significant.Statistical Package for Social Sciences  was used for data analysis. The normality of the information was analyzed using the Kolmogorov–Smirnoff test. Variables with normal distribution are presented as mean ± standard deviation (SD), or as median and first and third interquartile ranges (IQR). Frequencies are presented as percentages. Mann–Whitney U-test, Kruskal–Wallis test, and Dunn's post hoc test were used to analyze the differences among groups. To assess the value of IL-17A to discrimination of NMSC from the normal conditions, the area under the ROC curve (AUC) was determined and the best cut-off point for this marker as well as its sensitivity and specificity were calculated. In our study, N = 45, 75%) and the male-to-female ratio was 3 : 1 (45 : 15). The mean age of NMSC patients was 67.60 ± 12.82 years, and the most common diagnosis was SCC . Most of the lesions were located in the sun-exposed area 54 (93.10%) and most of them were below 4 cm (66.7%). None of the patients had metastatic lesions. In the present study, a total number of 60 NMSC patients and 57 age-sex matched healthy individuals, as the control group, were investigated. The majority of the patients were male (P < 0.001). In subgroup analysis according to pathologic diagnosis, serum levels of IL-17A were not different between SCC and BCC patients (P=0.793). However, patients with SCC had higher levels of IL-17A in their serum in comparison to the controls (5.95 (3.20–6.13) versus 3.20 (3.10–5.75) pg/ml; P < 0.001) (P=0.001) (P=0.286) or sex (P=0.357) of the patients. There was no significant difference in serum levels of IL-17A in NMSC patients with sun-exposed lesions and patients with nonsun-exposed lesions (P=0.124). Furthermore, no relationship was found between number of lesions (single or multiple), tumor size, and IL-17A serum levels  (The median levels of IL-17A in the NMSC patients were 5.89 (3.20–6.13) pg/ml and in the control group was 3.20 (3.10–5.75) pg/ml respectively, which indicated significant differences (< 0.001) . InvestiP=0.001) . In thisctively) . Accordictively) , the meaα, thereby eliciting an inflammatory response in patients. They suggest that increased Th17 counts and IL‐17 levels are potential biomarkers for poor prognosis in ovarian cancer [Skin cancer is one of the most important cancers worldwide that imposes great economic and medical burdens. A conclusive link between inflammation and skin cancer has been demonstrated . Inflammn cancer . In anotn cancer .α can induce the production of cytokines necessary for tumor progression [κB and STAT3 are fundamental for the development of inflammation-promoted cancer, such as skin cancer [κB in immune cells has been shown to result in the expression and production of multiple proinflammatory cytokines such as IL-17 which promotes the development of cancer [In contrast with different kinds of malignancy, less examinations have been performed on serum levels of IL-17A in skin cancer, especially NMSC. Nardinocchi et al. have indicated that both SCC and BCC are penetrated with a high number of IL-17+ T lymphocytes. They also found that IL-17 can increase the proliferation and migration of the BCC and SCC cell lines and that IL-17 alone or in combination with TNF-gression . As a magression –25. NF-κn cancer , 27. In f cancer , 29. Wanf cancer , 30. Ultf cancer . Exposurf cancer . Studiesf cancer .The immune system functions contradictory, which can either facilitate or inhibit the growth of cancer. The definitive aim for cancer immunology research is to achieve a treatment outcome in which IL-17A and other cancer-promoting cytokine signals are blocked to reduce the growth of cancer cells and weaken resistance to therapy and their survival , 35. IL-According to our results, it can be concluded that IL-17A may play a substantial role in inducing the growth and progression of NMSC. Furthermore, IL-17A may be used as a therapeutic target in NMSC patients in the future, so to determine this effect, more studies are needed.


This study aims at providing estimates on the transmission risk of SARS-CoV-2 in schools and day-care centres. We calculated secondary attack rates (SARs) using individual-level data from state-wide mandatory notification of index cases in educational institutions, followed by contact tracing and PCR-testing of high-risk contacts. From August to December 2020, every sixth of overall 784 independent index cases was associated with secondary cases in educational institutions. Monitoring of 14 594 institutional high-risk contacts (89% PCR-tested) of 441 index cases during quarantine revealed 196 secondary cases . SARS-CoV-2 infection among high-risk contacts was more likely around teacher-indexes compared to student-/child-indexes (incidence rate ratio (IRR) 3.17, 1.79–5.59), and in day-care centres compared to secondary schools , mainly due to clusters around teacher-indexes in day-care containing a higher mean number of secondary cases per index case (142/113 = 1.26) than clusters around student-indexes in schools (82/474 = 0.17). In 2020, SARS-CoV-2 transmission risk in educational settings was low overall, but varied strongly between setting and role of the index case, indicating the chance for targeted intervention. Surveillance of SARS-CoV-2 transmission in educational institutions can powerfully inform public health policy and improve educational justice during the pandemic. The SARS-CoV-2 pandemic urges government leaders to define priorities when implementing anti-epidemic measures in public domains. This task requires a profound scientific basis when balancing the obvious benefits of anti-epidemic measures against their hazards on the social, economic and health sector. While the educational and psychological impact of school closures on child health is rather well studied –3, our kThe present study provides estimates of the SARS-CoV-2 transmission risk in schools and day-care centres during exponentially increasing COVID-19 population incidence from August to December 2020 in Germany, i.e. before the advent of SARS-CoV-2 variants of concern. Due to the large sample size, we also present analyses on the variation in transmission risk by a variety of contextual characteristics of the index case.The presented data were collected in Rhineland-Palatinate, one of the 16 Federal States of Germany with an overall population of about 4.1 million, 1492 schools, 406 607 school-children and 144 245 children below 6 years of age in day-care . We repoThe 24 District Public Health Authorities (DPHAs) in Rhineland-Palatinate are responsible for all investigations around notifiable diseases, and represent populations between 61 000 and 430 000 individuals per district. Following the identification and statutory notification of a COVID-19 case, qualified personnel at the competent DPHA interviews this index case, tries to investigate the source of infection (primary case), traces contacts, and initiates a quarantine and active follow-up in those at high risk of transmission (category-I contact) for 14 days after the last contact to the index case. A category-I contact was defined as a person who either stayed face-to-face (<1.5 m) with a COVID-19 case for 15 min or longer, or in the same room (i.e. irrespective of distance) for 30 min or longer . AccordiAt the start of the study, a two-page data form was distributed to all DPHAs, alongside with instructions on inclusion and exclusion criteria for index cases and a link to upload completed data forms (ec.europa.eu/eusurvey). For inclusion into this study, an index case was defined as an individual that (i) tested positive for SARS-CoV-2-RNA from respiratory material, (ii) was notified as working in or attending an educational institution, and (iii) had worked in or attended the institution for at least one day during the infectious period. The infectious period was defined as follows: (i) for symptomatic index cases as time from 2 days before to 10 days after onset of symptoms; (ii) for asymptomatic cases with unknown origin from 2 days before to 10 days after the date of taking the diagnostic swab; and (iii) for asymptomatic cases with known contact to a primary case from 3 days to 15 days after exposure.A secondary case was defined as an individual that (i) was identified as a category-I contact person to an index case by the competent DPHA, (ii) tested positive for SARS-CoV-2-RNA during the quarantine associated with that index case, and (iii) was unlikely a co-primary case, based on evidence on the assumed chain of infection that evolved during the competent DPHA's investigation. Contact persons and secondary cases not attending the educational institution, e.g. persons living in the same household, were not to be reported in this data form. Beginning with 17 August 2020, we asked DPHAs to file one data form for each eligible index case about 2 weeks after its identification, when information on all potential secondary cases would be available, i.e. after completion of quarantine of the contact persons.P-value testing H0: ‘both SARs are equal’ (IRR = 1.0). For sensitivity, we repeated these analyses based on the number of PCR-tested contacts only.Assuming equal exposure time in all contact persons of a given index case, we calculated secondary attack rates  as the proportion of secondary cases among high-risk contact persons of a given index case during a 14-day period following the last contact (i.e. quarantine), together with corresponding binomial 95% confidence intervals (95% CIs), taking account of the clustering of contact persons in index cases . AssociaP-values testing H0: RR = 1.0 using binomial regression. Since this part of the analysis did not rely on an individual contact person denominator, it also included data from index cases with missing information on the total number of contact persons and/or the number of PCR tests done.The risk of causing a SARS-CoV-2 cluster (in brief ‘cluster risk’) was calculated by dividing the number of index cases that caused one or more secondary cases by the total number of observed index cases. To compare cluster risks by exposure category, we estimated risk ratios (RRs) and their 95% CIs and Finally, we compared the mean number of secondary cases per index (MSPI) among children/students and teachers, associated with index cases in students/children and in teachers in different institutions, respectively. As with the SARs, we used negative binomial regression models to compare differences in the count of secondary cases per index case by exposure category. All analyses and models accounted for clustering of secondary cases by index case and were conducted using Stata SE version 16.1 and SAS version 9.4 , 23.The collection, analysis and communication of the presented data take place in response to the global COVID-19 pandemic and are mandated by the German Infectious Diseases Protection Act. Ethical approval was waived by the competent ethics committee, Federal State Medical Council (Landesärztekammer) in Rhineland-Palatinate, Mainz, Germany (application no. 2021-15634-r).The course of the COVID-19 pandemic in Rhineland-Palatinate was comparable to all of Germany and was characterized by an exponential increase from the end of September  until the end of October (cw43), a further growth at a lower rate (cw44–45), followed by a fluctuation on a high level of about 5000–7000 new cases per week (cw46–52), which equals a 7-day incidence rate of 120–170 per 100 000 . SixteenOverall, DPHAs provided information for a total of 784 independent notified index cases attending an educational institution prior to diagnosis of a SARS-CoV-2 infection . There were 130 clusters of secondary cases reported via the SARS-Surveillance  , right. P < 0.001), symptom status at the time of diagnosis , type of institution  and age (older than 35 years > age between 6 and 21 years)  , left.P < 0.001) (P < 0.0001). A similar comparison looking at secondary cases in children/students found a similar but less pronounced difference towards student/child-secondaries being more likely identified in clusters around teacher-indexes  compared to those around students/children-indexes . Looking at role-specific cluster composition while stratifying by type of institution identified on average 1.26 MSPI around teacher-indexes in day-care, roughly evenly divided between teacher-secondaries (0.66 MSPI) and children-secondaries (0.59 MSPI). In schools, about 0.50 MSPI were identified around teacher-indexes, mostly in student-secondaries (0.44 MSPI), while teacher-secondaries were rare (0.06 MSPI). We observed on average two secondary cases per three child-indexes in day-care institutions (0.66 MSPI), which were about evenly divided between child-secondaries (0.38 MSPI) and teacher-secondaries (0.28 MSPI). Student-indexes in schools had the lowest MSPI: on average only one secondary per six student-indexes (0.17 MSPI). Among these, we hardly ever observed teacher-secondaries (0.004 MSPI).Teacher-indexes were associated with on average more secondary cases  = 1.08) than students/children  . Assessivs. 1.9) explaining why ‘black dots’ prevail when moving from the lower left to the upper right corner of the grid in The 329 secondary cases reported in this study occurred in 130 clusters, while the majority, 654 of overall 784 indexes (83%), were associated with zero secondary cases. In those 130 cases, where transmission occurred, the average cluster size was 2.5 secondary cases . There were nine clusters reported with seven or more secondary cases , of whicThis study provides evidence for an overall low SARS-CoV-2 transmission risk in educational settings from August to December 2020 in Germany, a period with sharply increasing incidence rates, but with preventative measures in place. We found that approximately 1.3% of school contacts of an index case classified as category-I became SARS-CoV-2-positive. When restricting the denominator to PCR-tested contacts, we estimated a comparable SAR of 1.5%. These numbers are well in line with other published findings on the risk of transmission in schools from Australia, Germany, Italy, Ireland and Singapore, where several smaller studies found comparable SARs between 0% and 3% –8, 24.Compared to student-/children-indexes, we found that the SAR was higher when the index case was a teacher. Likewise, the cluster risk and the mean number of secondary cases were higher when teachers were identified as index cases compared to students. Although not formally tested in other studies, mainly due to the small sample size, descriptive findings in published literature already point towards larger numbers of secondary cases in teacher-index cases and support our findings , 25–27. vs. symptomatic cases [The role of asymptomatic cases in the spread of the COVID-19 pandemic has been a subject of an ongoing debate. A review and meta-analysis found that the proportion of asymptomatic cases was 17% of all COVID-19-positive cases , 29. Theic cases .vs. 329/784 = 0.42 and 196/81 = 2.4 vs. 329/130 = 2.5, respectively (see This study has limitations. First, we do not have follow-up data of all notified cases in the context of educational institutions from all 16 reporting DPHAs. This raises the question of selection bias. However, we advised DPHAs to report consecutive index cases over at least a 4-week period or longer, thus reducing the chance of systematic under- or over-reporting of more or less salient index cases and associated under- or overestimation of transmission risk. Second, although all DPHAs routinely offer PCR tests to all contact persons to a COVID-19 index case at high risk of transmission in the educational setting, 44% of our sample came from DPHAs that had outsourced sampling and testing to community testing centres. From these DPHAs, we received reliable information on secondary cases, but not on total contact persons and contact persons tested, since only positive test results are notifiable by testing centres and associated laboratories. However, considering the similarity of the average number of secondary cases and average cluster size in both samples 196/441 = 0.44 vely see , we are A third limitation concerns the quality of information that allows the DPHA to identify the underlying chain of transmission and thus to judge whether the index case was indeed the primary case in the given setting. Hypothesizing that some index cases were included although not being the primary case and knowing that children are more likely to have asymptomatic infection compared to adults, this may have led to an overestimation of the risk of transmission around teacher-secondaries through increasing the risk of misclassifying children as secondary cases. Although the presented risk and MSPI ratios may to a limited extent be an overestimation of the true difference between teachers and children/students, it seems unlikely that misclassification explains the whole magnitude of the difference observed in our data. This seems particularly true since the proportion of ‘entirely asymptomatic cases’, even in small children, is a function of how the investigation was performed and tends to be substantially higher in settings where superficial investigations are used , 29. TheFourth, our results may be limited by representing the transmission situation in the educational setting at times, where literally no SARS-CoV-2 variants of concern have been present in Germany, yet. Although some of these have been shown to be associated with higher transmissibility rendering the absolute estimates presented outdated, our findings on transmission patterns and relative differences on transmission by setting and role are likely to be independent of strain type and thus to hold true in the presence of variants of concern as well.Finally, our study attributes all transmissions detected around COVID-19 indexes in the educational setting to transmission in schools or day-care. This approach does not acknowledge that children/students and teachers may also have contact with each other outside the institution, e.g. during leisure activities. This may have increased the presented risk estimates, particularly for child-to-child transmission, where exposure in the classroom and during leisure commonly coincides.During a period of sharply increasing COVID-19 incidence rates in the population from August to December 2020, we found a low and rather stable transmission risk in schools, which provides evidence for the effectiveness of the preventative measures in place in German educational institutions. We found evidence for an increased risk of SARS-CoV-2 transmission to high-risk contact persons to teacher-indexes in day-care centres, mainly due to a stronger association of teacher-indexes with teacher-secondaries. Much less SARS-CoV-2 infection, by contrast, was found among high-risk contact person to children-/student-index cases and only a negligible number of infections could be observed when school-teachers were close contacts to student-indexes. The early communication of the presented findings informed public health decisions in Germany and Europe –33. Cont


We developed TripletRes to deduce protein contact-maps from discretized distance profiles by end-to-end training of deep residual neural-networks. Compared to previous approaches, the major advantage of TripletRes is in its ability to learn and directly fuse a triplet of coevolutionary matrices extracted from the whole-genome and metagenome databases and therefore minimize the information loss during the course of contact model training. TripletRes was tested on a large set of 245 non-homologous proteins from CASP 11&12 and CAMEO experiments and outperformed other top methods from CASP12 by at least 58.4% for the CASP 11&12 targets and 44.4% for the CAMEO targets in the top-L long-range contact precision. On the 31 FM targets from the latest CASP13 challenge, TripletRes achieved the highest precision (71.6%) for the top-L/5 long-range contact predictions. It was also shown that a simple re-training of the TripletRes model with more proteins can lead to further improvement with precisions comparable to state-of-the-art methods developed after CASP13. These results demonstrate a novel efficient approach to extend the power of deep convolutional networks for high-accuracy medium- and long-range protein contact-map predictions starting from primary sequences, which are critical for constructing 3D structure of proteins that lack homologous templates in the PDB library.The topology of protein folds can be specified by the inter-residue contact-maps and accurate contact-map prediction can help  Ab initio protein folding has been a major unsolved problem in computational biology for more than half a century. Recent community-wide Critical Assessment of Structure Prediction (CASP) experiments have witnessed exciting progress on ab initio structure prediction, which was mainly powered by the boosting of contact-map prediction as the latter can be used as constraints to guide ab initio folding simulations. In this work, we proposed a new open-source deep-learning architecture, TripletRes, built on the residual convolutional neural networks for high-accuracy contact prediction. The large-scale benchmark and blind test results demonstrate competitive performance of the proposed methods to other top approaches in predicting medium- and long-range contact-maps that are critical for guiding protein folding simulations. Detailed data analyses showed that the major advantage of TripletRes lies in the unique protocol to fuse multiple evolutionary feature matrices which are directly extracted from whole-genome and metagenome databases and therefore minimize the information loss during the contact model training. PLOS Computational Biology Methods paper.This is a ab initio structure prediction) [Protein structure prediction represents an important unsolved problem in computational biology, with the major challenge on distant-homology modeling (or diction) ,2. Recendiction) –6.ab initio protein structure prediction is, however, not new, which can be traced back to at least 25 years ago [The idea of developing sequence-based contact-map prediction to assist ears ago ,8. In geears ago  and covaears ago , to predears ago –16 is stAs a more general approach, MLMs intend to learn the inter-residue contacts from sequential information and coevolution analysis features with supervised machine learning models trained with known structures from the PDB. Early attempts utilized support vector machines (SVMs) ,18, randhttps://zhanglab.ccmb.med.umich.edu/TripletRes/.In this work, we proposed a new deep learning architecture, TripletRes, built on a residual neural network protocol  to integhard by CAMEO [To examine the contact prediction pipelines, we collected two independent sets of test targets, including 50 non-redundant free-modeling (FM) domains from the CASP11 and CASP12 and 195 non-redundant targets assigned as by CAMEO . Tripletby CAMEO . Here, nL/10, L/5, L/2, and L of medium- (12≤|i–j|≤23) and long-range (|i–j|≥24) contacts are evaluated, where i and j are sequential indexes for the pair of considered residues and L is the sequence length of the target. We focus on the performance of FM targets (or hard targets in CAMEO) and on long-range contacts for evaluation, since the metric is most relevant for assisting the prediction of the tertiary structure of non-homologous proteins [Following the CASP criterion , two resproteins ,33.L/10, L/5, L/2, and L predicted contacts by TripletRes is 55.1%, 53.2%, 57.1%, and 58.4% higher, respectively, than the precision achieved by DeepContact, the most accurate third-party program in this comparison, which correspond to statistically significant p-values of 4.1e-08, 2.5e-07, 4.4e-10, and 1.1e-11 in the Student’s t-test. Notably, TripletRes only uses coevolutionary features, which is a subset of the diverse features employed by DeepContact. The better performance is also probably due to the more effective integration of raw coevolutionary information in the TripletRes neural-network training.L precision of TripletRes is 36.9% higher than that of ResPRE with a p-value of 1.9e-07 on 50 FM targets. ResPRE achieved a significantly higher precision on CAMEO than the FM dataset, but its precision is still lower than that of TripletRes. For example, the mean precision of the top-L long-range contacts by TripletRes is 12.6% higher than that of ResPRE on the CAMEO targets. Given that both programs utilized the same precision matrix feature, the superiority of TripletRes is mainly attributed to the integrations of triplet coevolutionary features. In addition, as examined in detail below, the supervision of the distance predictions and the new deep MSA constructions also helped improve the accuracy of the TripletRes models.TripletRes also outperforms ResPRE, an in-house program previously trained on precision matrix , by a laL precision increases from 33.9% to 42.9% on 50 CASP FM targets. Nevertheless, TripletRes still take the lead over the control methods and the top-L precisions on CASP and CAMEO targets are 28.8% and 27.9% higher than those of the best third-party programs, DeepContact.The proposed TripletRes pipeline’s performance could be overrated since more data have been used compared to those methods in CASP11&12. To reduce the bias, we have ensured that the maximum pairwise sequence identity is 30% between training and test set. In addition, we have tweaked those control methods by replacing their MSAs with DeepMSA and Feature extraction is essential for all machine-learning based modeling approaches. To quantitatively examine the effectiveness of the feature extraction strategy and the contribution of different feature types in TripletRes, we compare in L/5 contact prediction are 84.2%, 87.5%, and 88.6%, respectively, for COV, PRE, and PLM features. If the post-processed features of Eqs A and B in It can be observed from One reason for the performance degradation by the post-processing approach is that the potential score for different types of residue-pairs have been treated equally and the sign of these potential scores is thus completely ignored in Eq A in L/5 precisions of the predictors trained by three individual component features and their ensemble on the validation set containing 767 sequences. Note that the feature ensemble model in Compared to ResPRE , a majorL/5 and top-L precisions, excluding the PLM feature has the lowest values during the training process, indicating that the PLM feature makes significant contributions. Interestingly, feature sets without PRE or COV feature and feature ensemble seem to be indistinguishable for top-L/5 precision. While for top-L precision, the full TripletRes with the triplet features ensemble stands out, achieving a precision of 68.2%, higher than the precisions of 67.3%, 67.5%, and 67.4% without COV, PRE, and PLM feature respectively. Surprisingly, COV and PRE seem to have similar contributions to the TripletRes model, even though the model using only PRE feature is previously shown to significantly outperform the model using only COV feature . Typically, with a single distance threshold of 8 Å, such a loss function does not encode detailed distance information, e.g., residue pairs separated by 9 Å will be treated the same as those by 22 Å. Alternatively, recent methods ,35,37 haL/5 and top-L precisions between TripletRes programs trained with L/5 precision from 68.7% to 71.4%, and 74.1% to 75.6%, for the CASP set and CAMEO set, respectively, which correspond to a p-value of 2.1e-02 and 7.9e-03 in Student’s t-test. Interestingly, when more top-ranked contacts are considered , the p-values become more significant and decrease to 1.6e-04 and 2.0e-07 on the two datasets, respectively, which means the distance information may have a stronger effect on improving the precision when more contacts are evaluated. Protein structure prediction methods can thus benefit more from TripletRes, which was trained with the discrete distance loss function because more predicted contacts can be reliably considered as restraints for protein folding.L/5 and top-L precisions with different loss functions on the different fold types, with median and mean precisions marked in solid and dash lines, respectively. Structures from 195 CAMEO set and CASP 11&12 set are classified into 63 alpha proteins, 24 beta proteins and 157 alpha&beta  proteins. For three fold types, consistent improvements can be observed with the distance loss function for all evaluation indexes. For example, for long rang top-L predicted contacts, training with discrete distance loss function achieves precisions of 34.9%, 49.0%, and 54.0% for alpha, beta, and alpha&beta folds, which are slightly higher than the baselines, corresponding to p-values of 4.0e-03, 2.0e-02 and 9.4e-08, respectively. Among three types of fold types, alpha proteins have the lowest mean top-L/5 and top-L precision, regardless of the loss function type; this may be due to the fact that contact patterns, including hydrogen-bonds, between alpha-helical segments are not as evident as those between beta-strand elements in proteins.To have a detailed analysis of the effect of discrete distance loss function on different fold types, TripletRes utilizes MSAs as the only input and the quality of the latter is thus essential to the final contact prediction models. It is worth noting that the TripletRes model is trained on features extracted from MSAs generated by HHblits, but a deeper MSA generated by multiple databases has been used for test proteins . We expect the strategy could reduce over-fitting between the training and test proteins.L prediction by each dataset. For the CASP FM targets, the usage of deep MSAs during testing significantly improves the mean precision of TripletRes from 40.0% to 46.4% with a p-value 1.9e-05 in Student’s t-test, where 35 out of 50 FM targets (70%) achieve a higher precision with deep MSAs while only 8 targets (16%) do so when the HHblits MSAs are used. The same trend can be observed in the CAMEO targets, where the p-value of improvement in long-range top-L precision is 1.7e-06. This difference is mainly due to the higher number of homologous sequences collected in deep MSA search protocol, which allows the extraction of more reliable coevolutionary information. For example, the average number of effective sequences of MSAs, or Neff calculated by To examine the impact of different MSA collections on the contact models, L contact predictions. The deep MSA search increased the Neff value to 3.78, where the number of true contacts with the deeper MSA increases to 73, which is 87.2% higher than that with the HHblits MSA. In L long-range predictions; while 42 more contacts are detected by TripletRes through the deep MSA that has a Neff of 14.0. These examples highlight again the importance of using deep MSA pipeline for coevolutionary feature collection and the impacts on final contact-map prediction. The new contacts correctly predicted after performing deep MSA searching strategy are marked as dashed lines in In http://www.predictioncenter.org/casp13/zscores_rrc.cgi), with another top method RaptorX-Contact which also ranked as the top method in previous CASPs. In L/5 contacts on the 31 FM targets, TripletRes_CASP13 achieved a mean precision of 64.6%, while the mean precision of RaptorX-Contact, DMP, and ZHOU-Contact are 69.4%, 60.2%, and 58.3%, respectively. TripletRes, however, achieves the highest precision of 71.6% for long-range top L/5 contacts. Here, TripletRes and TripletRes_CASP13 are based on the same input MSAs and the only difference between them is that TripletRes utilizes a new loss function , benefited from the distance information during the training. As shown in -L/5 correctly predicted long-range contacts, 94.7% of them have the distance profile with a probability peak at <8Å and nearly 74% of the residue pairs have the accumulated probability >80% in the region below 15Å, indicating a high confidence of contact prediction on the residue pairs based on the distance profile.In L/5 precision of 76.2% on the 37 test sequences, 9.2% higher than that of TripletRes with a p-value of 1.7e-03. Such differences in performance with different amounts of training data indeed demonstrate the importance of available dataset when training the model. Compared to TripletRes, trRosetta has a slightly higher prediction along all the cutoffs; the difference is however statistically insignificant, with the p-value equal to 0.68, 0.59, 0.15, and 0.20 for top-L/10, L/5, L/2 and L precisions, respectively. It is noted that the higher contact accuracy by trRosetta is mainly attributed to various auxiliary prediction tasks such as orientation prediction, while for TripletRes, the improvements mainly come from the ensemble of multiple co-evolutionary features. In this sense, the proposed TripletRes method should be considered complementary to the trRosetta.Note that both TripletRes_CASP13 and TripletRes are trained on the same training set before CASP13. To examine the impact of the size of training dataset on the proposed framework, we re-train the TripletRes model with a dataset newly collected after CASP13 with 26,151 PDB sequences and perform the evaluation on a test set containing 37 sequences , with thL long range contact precision has reportedly achieved 46.1% [Apart from trRosetta and DMP discussed above, AlphaFold  also pered 46.1% , which wThe re-training of the TripletRes (Post-CASP13) model took up to 30 days on 4 Nvidia P100 GPUs from the public XSEDE Comet Cluster  due to tProtein contact-map prediction has been critical to assist protein folding in the form of spatial constraints. This work presented a new deep learning method for high-accuracy contact prediction by learning from raw coevolutionary features extracted with deep multiple sequence alignments. The method was tested on FM domains in CASP11-13 and hard targets from CAMEO experiments, which demonstrated the effectiveness of the proposed method.Several factors were found to contribute to the success of the TripletRes pipeline. First, coupling deep residual convolutional networks directly with raw coevolutionary matrices can result in better performance than feeding neural networks with the post-processed features. Second, a triplet of coevolutionary features, from covariance matrix, inverse covariance matrix and the inverse Potts model approximated by pseudolikelihood maximization, are ensembled in TripletRes by a set of four neural networks constructed with residual blocks. This feature ensemble strategy was found to enable more accurate prediction than using the three sets of features individually. Third, including more discrete distance information into the network training was proven to be beneficial to the contact-map prediction compared to binary contact training, although the contact-map models are binary on their own. This is largely because the distance-based loss function enables the learning of detailed spatial features specified by the sequence profiles. Finally, a hierarchical sequence searching protocol was proposed to obtain deeper MSAs, which impact the performance of the final model prediction. A significant improvement of contact prediction precision can be achieved through MSAs generated by searching an enlarged protein sequence database. These data underscore the impact of the volume of the sequence database on contact/distance prediction. The studies extending the DeepMSA pipeline to utilize the enlarged databases are in progress.ab initio 3D structure construction, where a significant amount of efforts has been made along this line in the past decades [It is worth noting that the major goal of contact-map prediction is for assisting  decades ,33,40–42 decades ,35, contTripletRes is a deep-learning based contact-map prediction method consisting of three consecutive steps . It firsTo help offset the overfitting effects, TripletRes creates MSAs using different strategies for training and testing protein sequences. For training proteins, MSAs are created by HHblits with an E-value threshold of 0.001 and a minimum sequence coverage of 40% to search through Uniclust30 (2017_10)  databaseN is the total number of sequences in the MSA, Sm,n between sequences m and n is over 0.8; or = 0 otherwise. To assist the MSA concatenation, the jackhmmer hits are converted into an HHblits format sequence database, against which a second HHblits search was performed. In case that Neff is still below 128, a third iteration is performed by hmmsearch [For test proteins, the DeepMSA pipeline  was utilmmsearch  through mmsearch , where tfi(a) is the frequency of a residue a at position i of the MSA, fi,j is the co-occurrence of two residue types a and b at positions i and j.Three sets of coevolutionary features are extracted from the deep MSAs. First, the covariance (COV) feature measures the marginal dependency between different sequential positions and is calculated bytr(SΘ) is the trace of matrix SΘ; log|Θ| is the log determinant of Θ; and ρ = e−6 being a positive regularization hyper-parameter.The COV feature captures marginal correlations among variables, which contains transitional correlations. The negative of the inverse of the covariance matrix, i.e., precision matrix, can be interpreted as the Mean-field approximation of Potts model  and thusThe last feature, which was firstly introduced by plmConv , is the P(σm) is the probability model for the m-th sequence in the MSA and l-th position in the sequence byh and J are single site and coupling parameters, respectively. In TripletRes, the raw coupling parameter matrix J is used as the PLM feature.Here, L by 21*L matrix for a protein sequence with L amino acids. The entries of the 21 by 21 sub-matrix of a corresponding amino acid pair are the descriptors, which are fed into a convolutional transformer as conducted by a fully convolutional neural network with residual architecture   as the dThe activation function of the last layer is a softmax function which outputs the probability of each residue pair belonging to specific distance bins. Here, the residue-residue distance is split into 10 intervals spanning 5-15Å with an additional two bins representing distance less than 5Å and more than 15Å, respectively. The whole set of deep ResNets are trained by the supervision of the maximum likelihood of the prediction, where the loss function is defined as the sum of the negative log-likelihood over all the residue pairs of the training proteins:T is the total number of residue pairs in the training set. t-th residue pair of native structures falls into k-th distance interval; otherwise t-th residue pair falls into the k-th distance interval. The probability of the t-th residue pair forming a contact Pt is the sum of the first 4 distance bins:Here, The training process uses dropout to avoid over-fitting, where the dropout rate is set to 0.2. We use Adam , an adapS1 Fig(a) top-L/5 precision, (b) top-L precision.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Tablep-values in parenthesis are from a Student’s t-test between TripletRes and each of the control methods, where bold fonts highlight the best performer in each category.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S3 Text(PDF)Click here for additional data file.S4 Text(PDF)Click here for additional data file.S5 Text(PDF)Click here for additional data file.


Bisphenol A (BPA), the most common endocrine-disrupting chemical, has been associated with male reproductive dysfunctions. Recently, it has been shown that BPA may also affect miRNAs expression. Herein, we aimed to evaluate the association of BPA levels with steroid hormone concentration and circulating miRNAs levels to investigate the potential direct effect of BPA on homeostasis in the testis environment. The level of BPA in the seminal plasma of azoospermic men was significantly higher compared to the healthy control. The concentrations of estradiol (E2) and androstenedione (A) were significantly decreased in the seminal plasma of azoospermic men compared to the normospermic men. The levels of miR-let-7a, miR-let-7b, and miR-let-7c were significantly up-regulated, and the level of miR-518f was significantly down-regulated in the seminal plasma of the azoospermic men compared to the healthy control. The level of BPA correlated negatively with sperm concentration and normal semen morphology. A significant positive correlation was found between BPA levels and miR-let-7a and miR-let-7c levels, whereas BPA negatively correlated with miR-518f levels. Our results suggest that BPA may negatively affect sperm quality. Moreover, BPA correlated with the miR-let-7a, miR-let-7c, and miR-518f levels in seminal plasma, which suggests that BPA may act directly in seminal plasma, affecting the testicular environment. Bisphenol A (BPA) is the most common industrial plasticizer classified as an endocrine disrupting-chemical (EDC) . BPA hasBPA may alter the function of the hypothalamic–pituitary–testicular axis and cause testicular dysgenesis and atrophy, enlargement of the prostate gland, and changes in semen parameters ,10,19. IMiRNAs are non-coding, single-stranded RNA molecules composed of 18–24 nucleotides . They arAlthough the effect of BPA on reproductive tissues is the topic of recent extensive research, there are limited data on how BPA affects the reproductive tissues environment. Seminal plasma is a unique body fluid that creates the environment in which the effects of BPA may be expressed directly in testicular tissue. Herein, we aimed to evaluate the association of BPA levels with steroid hormone concentration and circulating miRNAs levels to investigate the potential direct effect of BPA on homeostasis in the testis environment. The miRNAs chosen for this study were miR-let-7a, miR-let-7b, miR-let-7c, and miR-518f as they have been associated with estrogen and androgen signaling ,36. Moren = 20), oligoasthenoteratozoospermia (n = 46), and control normospermic (n = 50) were enrolled in this study. The mean age and BMI of the men were similar in all groups  in accordance with WHO guidelines ). WHO laboratory manual for the examination and processing of human semen, 5th ed. World Health Organization. A minimum of 200 sperm cells from at least four different fields were analyzed from each specimen. Each sample was assessed twice. Measured semen parameters included semen volume, sperm concentration, total sperm count, and sperm motility. Sperm morphology was evaluated using the Diff-Quik staining set . Total sperm count (106) was calculated by multiplying sperm concentration (106/mL) by semen sample volume (mL).The semen samples were obtained in a private room after a recommended 2–5 days of sexual abstinence. Semen analyses were conducted after liquefaction at 37 °C for 30 min by the micro-cell slide and the computer-aided semen analysis . Deuterated BPA was used as an internal standard. The BPA concentrations were calculated using a calibration curve for concentrations from 0 to 4.3 nmol/L.Bisphenol A was measured from seminal plasma samples that were thawed at room temperature. BPA was extracted from the samples three times, using sonication in acetonitrile and vortex mixing. The collected supernatants were first centrifuged for 10 min at 5000× Estradiol, progesterone, and testosterone levels were measured by the immunoanalyzer Cobas e411  using the Elecsys Estradiol III Gen Kit , the Elecsys Progesterone II Gen Kit , and the Elecsys testosterone II Gen Kit . Androstenedione concentrations were measured by the immunoanalyzer IMMULITE 1000  using the IMMULITE 1000 Androstenedione kit .C. elegans cel-miR-39 . The synthetic cel-miR-39 was reconstituted in nuclease-free water , resulting in a 2 × 1010 copies/μL stock. Next, the working solution was prepared to provide 1.6 × 108 copies/μL solution. To all samples, 3.5 μL of the cel-miR-39 working solution was added. The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer , while its quality was verified by denaturing agarose gel electrophoresis.The total RNA was isolated from the seminal plasma using the TRIzol extraction method . Three purification steps were added. To achieve technical normalization, all samples were spiked with synthetic non-human ®MicroRNA Reverse Transcription Kit . The reaction was performed with 5 ng of total RNA in a total volume of 15 μL. The mixture was incubated at 16 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min as recommended by the manufacturer. TaqMan miRNA probes  were used to assess the selected miRNA expression profile. Real-time PCR assays were performed in a total of 20 μL reaction volume with TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems). Each sample was processed in doublets with an initial denaturation at 95 °C for 10 min followed by 40 amplification cycles at 95 °C for 15 s and 60 °C for 60 s using the 7500 Real-Time PCR System (Applied Biosystems). The miRNA Ct-values were normalized using the exogenous cel-miR-39 and endogenous U6 snRNA. After the reactions, the Ct data were determined using default threshold settings and the mean Ct was determined from the duplicate PCRs. The expression of miRNA relative to U6 snRNA was determined using the 2−ΔCt method.cDNA synthesis was performed using the TaqManp < 0.05.Statistical analyses were conducted using GraphPad Prism 9 . Due to non-normal distribution of data, the non-parametric Kruskal–Wallis test with Dunn’s post hoc test was applied in the analysis. The correlations between the studied parameters were assessed with Spearman’s correlation coefficient. Statistical significance was assumed at No significant differences in age and BMI were observed between men with oligoasthenoteratozoospermia, men with azoospermia, and the control . The levThe level of E2 was significantly decreased in the seminal plasma of the group of men with azoospermia compared to the control a. There We observed higher levels of circulating miR-let-7a, miR-let-7b, and miR-let-7c in the seminal plasma of the group of men with azoospermia compared to the control a–c. No dr = −0.232, p = 0.01, r = −0.22, p = 0.02 and r = −0.193, p = 0.04; respectively)    . Significtively) . The lev = 0.01) . No corr = 0.01) .BPA exposure has been associated with adverse effects on male reproductive functions, spermatogenesis, and fertility ,38. In tSince estrogens play an important role in testicular spermatogenesis, several studies have investigated the impact of BPA on estrogen levels and estrogen metabolism ,13,38. ARecently, aberrant miRNAs expression in testicular tissue has been reported . AlteratIn conclusion, our findings showed that BPA may have different concentrations in seminal plasma and may negatively correlate with sperm quality parameters. It seems that BPA may act directly in seminal plasma, thus affecting the testicular environment and the levels of hormones and miRNAs. However, the exact direct mechanism of BPA action and its interaction with miRNAs in seminal plasma requires further investigation.